The objective of the proposed studies is to solve the three-dimensional structure of the intact psi-site of Human Immunodeficiency Virus type 1 (HIV-1), the approximately 120 nt segment of RNA responsible for genome recognition, packaging, and virus assembly. The complete psi-complex includes two copies of psi-RNA and an undetermined number of units of nucleocapsid protein p7 (NC) and is too large to be structurally characterized by NMR. Its apparent flexibility has thus far prevented crystallization for structural studies by X-ray diffraction. The 3D structures of the 4 individual RNA stem-loops believed to form the psi-site and those of their complexes with NC have been solved by NMR and could be used as starting data for the molecular modeling of the intact complex. Distance constraints, spatial orientation, and points of contact between the stem-loops will be obtained by a novel strategy based on chemical and biochemical probes in combination with electrospray ionization-ion cyclotron resonance (ESI-ICR) mass spectrometry. In particular, we will apply ESI-ICR to obtain the stoichiometry of binding between psi-RNA and NC in the intact psi-complex and to characterize the products of nucleic acid and proteic nature (or both for crosslinked species) provided by probing psi-RNA and its complex with NC. Mass mapping and MS/MS sequencing techniques will be employed to identify the location of the points of attack of each probe. The distance constraints obtained experimentally will be used in restrained structural calculations to determine the global fold of psi-RNA in presence and absence of NC. The use of electrospray ionization mass spectrometry as the detection method for classical probes offers the possibility of detecting in parallel a large number of molecular contacts with no need for labor-intensive separation procedures or radioactive tracers. Based on the large choice of available probes and the capability of handling large size RNAs, which are beyond the detection limit of NMR spectroscopy, this approach promises to have a major impact on the investigation of RNA-RNA and RNA-protein interactions. The long-term goal is to understand the molecular basis for the recognition and packaging of the retroviral genome mediated by the psi-site. A full understanding of this mechanism is crucial to elucidate at the molecular level the process of assembly and maturation of HIV-1 and similar retro-viruses and should facilitate the design of new therapeutic strategies for AIDS and cancer.